Intra- and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cytoplasm from Raphanus sativus and Raphanus raphanistrum

In order to gain a better understanding of the evolution of Ogura male-sterile cytoplasm in radish, a large-scale sequence analysis of mitochondrial orf138 was conducted using 107 Japanese wild radishes, 29 cultivated radishes and seven Raphanus raphanisturum. A single approximately 0.8-kb fragment containing the orf138 locus was amplified from each plant by PCR, and the nucleotide sequence of an entire coding region of orf138 was determined by direct-sequencing procedures. An identical sequence to the published orf138 (Type A) was identified in Japanese wild radish, including a single plant in a population near Kagoshima prefecture where Ogura (1968) first found ’Ogura male-sterile radish’. Thus, it was confirmed that the ’Ogura male-sterile cytoplasm’ was derived from Japanese wild radish, with a Type A orf138 sequence, growing in this area. A total of six nucleotide changes and a single insertion/deletion (indel) were found in orf138 from both wild and cultivated radishes. By a combination of mutations, the orf138 sequences of the 143 radish plants were classified into nine types. Based on the pattern of mutations and the distribution of orf138 variants, it was concluded that the orf138 variants are derived from Type B or C, after Ogura-type cytoplasm was introduced from R. raphanistrum into Japanese wild radish. Received: 19 December 2000 / Accepted. 26 January 2001     

Inhibition of chalcone synthase expression in anthers of Raphanus sativus with Ogura male sterile cytoplasm

Background and Aims

Expression of the mitochondrial gene orf138 causes Ogura cytoplasmic male sterility (CMS) in Raphanus sativus, but little is known about the mechanism by which CMS takes place. A preliminary microarray experiment revealed that several nuclear genes concerned with flavonoid biosynthesis were inhibited in the male-sterile phenotype. In particular, a gene for one of the key enzymes for flavonoid biosynthesis, chalcone synthase (CHS), was strongly inhibited. A few reports have suggested that the inhibition of CHS causes nuclear-dependent male sterile expression; however, there do not appear to be any reports elucidating the effect of CHS on CMS expression. In this study, the expression patterns of the early genes in the flavonoid biosynthesis pathway, including CHS, were investigated in normal and male-sterile lines.

Methods

In order to determine the aberrant stage for CMS expression, the characteristics of male-sterile anthers are observed using light and transmission electron microscopy for several stages of flower buds. The expression of CHS and the other flavonoid biosynthetic genes in the anthers were compared between normal and male-sterile types using real time RT-PCR.

Key Results

Among the flavonoid biosynthetic genes analysed, the expression of CHS was strongly inhibited in the later stages of anther development in sterility cytoplasm; accumulation of putative naringenin derivatives was also inhibited.

Conclusions

These results show that flavonoids play an important role in the development of functional pollen, not only in nuclear-dependent male sterility, but also in CMS.Key words: Chalcone Synthase, flavonoids, Ogura cytoplasmic male sterility, CMS, pollen, Raphanus sativus     

Phytochrome-regulated transfer of fructosidase from cytoplasm to cell wall in Raphanus sativus L. hypocotyls

The far-red absorbing form of phytochrome, P_fr, rapidly increases the rate of transfer of -fructosidase (E.C.3.2.1.26) from the cytoplasm to the cell wall in radish hypocotyls. Far-red light increases the level of enzyme in a particulate fraction: after two hours of light treatment, the particulate enzyme is associated almost exclusively with the endoplasmic reticulum. Transfer from the endoplasmic reticulum to the cell wall involves an incorporation into Golgi bodies and the plasmalemma: these membrane fractions were separated by centrifugation on a discontinuous sucrose density gradient and their degree of purity was determined by the use of known biochemical markers. With respect to -fructosidase, light controls, via P_fr: (1) the total amount, (2) the incorporation into the endoplasmic reticulum and (3) the transfer to the cell-wall. These three processes have different sensitivities to cycloheximide.Abbreviations -FFase -fructosidase - IDPase inosine diphosphatase - SGTase UDPG-sterol glucosyltransferase - NCRase NADPH-cytochrome c-oxydoreductase - NPA N-naphtylphtalamic acid - BSA bovine serum albumine     

萝卜单倍体培养

1 植物名称　萝卜(Raphanus Sativus),又名莱菔、芦菔. 2 材料类别　花药. 3 培养条件 (1)胚状体诱导:以改良的Keller为基本培养基,10%蔗糖、0.8%琼脂粉,pH 5.8,不附加任何激素.(2)诱导分化培养基:MS+ 0.2 mg·L-1 6-BA,2%蔗糖、0.8%琼脂条,pH 5.5.     

Effect of copper on peroxidase activity and lignin content in Raphanus sativus

Copper (Cu²⁺) significantly inhibits the growth of radish (Raphanus sativus) seedlings at the concentration of 1 μM. As far as the relationship between the growth of radish roots and peroxidase (POD) activity is concerned, the reduction of radish roots is correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise in lignin content in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (K_m of 57.9 μM) for syringaldazine and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 and 0.12 μM^–1 s^–1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment can be a good candidate for lignification in radish roots.     

Fluorescence analysis of picoliter samples

The fluorescence intensity of picoliter-volume samples was quantitated by taking samples and standards into a single siliconized capillary, fixing the capillary under the objective of a microscope-fluorometer, and defining an effective “fluorescence chamber” within the capillary by placing an imaging diaphragm in the emission path. Samples were then moved, by pneumatic control with an air syringe, within the capillary to this “fluorescence chamber.” A diaphragm in the excitation path limited the volume of sample excited. Fluorescence from all samples was thus directly determined under identical optical conditions. The co-efficient of variation of replicate measurements was 3%; carry-over from sample to sample within the capillary was less than 2%. A 3-pl sample containing 0.3 amol of sodium fluorescein (about 200,000 molecules) could be discriminated from the background; fluorescence intensity was linear with concentration for three to four orders of magnitude. Fluorescence intensities of NADH and an ammonia-o-phthalaldehyde-thiol adduct were also determined. Using this “fluorescence chamber” allowed straightforward scaling down of a fluorescence assay for urea in 20-pl samples, lowering the limit of detection to 10 fmol, three orders of magnitude below a previously reported microscale assay. This technique is applicable to many fluorescence assays used in studies of cell physiology, and should allow routine measurement of metabolites in individual cells or of enzymes in individual subcellular organelles.     

Thiocyanate content in relation to the quality features of radish Raphanus sativus L

Relationships between radish thiocyanate content and its dry weight, the content of sugar, protein, fibre, ascorbic acid, some minerals, the incidence of plant shooting, the firmness and pithiness of storage-roots, and the ratio of leaves to storageroot (wt/wt) were investigated. The analysis of linear correlation was based on numerous data from the 4-year field experiment with six radish cultivars and different sowing and harvest dates. The content of thiocyanate in radish roots was found to be positively correlated with their dry weight, and the content of total protein, crude fibre, and soluble sugar. A strong relationship was found between the content of thiocyanate and dry weight of radish leaves. The negative correlation between the thiocyanate content in the leaves and the firmness of storageroots and the positive correlation with their pithiness might indicate the translocation of this compounds into green plant parts during the ageing of root tissue. The root thiocyanate content and the percentage of shooting correlated significantly only in the case of Tokinashi. The closeness of relations between the ratio of leaves/storage-root and thiocyanate content, though in general small, was affected also by a cultivar. A similar effect was observed for the correlations between the thiocyanate contents in leaves and storage-roots.     

Myrosinase in Raphanus sativus L.

A previous paper reported an investigation of myrosinase inthe seeds, seedlings and the different organs of mature plantsof Sinapis alba. The enzyme distribution was related to thehistological localization of myrosin cells. The present paperreports the situation for Raphanus sativus. Differences in myrosinasepatterns have been found but the two taxa are similar in thatthere is good evidence for the presence of the enzyme in myrosincells and in other non-specialized cells. Key words: Raphanus, Myrosinase, Isoelectric focusing, Myrosin cells     

萝卜花粉的营养成分研究

萝卜花粉的营养成分测定表明,其蛋白质含量28.38%,总糖含量33.33%,氨基酸总量27.59%,总黄酮含量3.57%,维生素和矿质元素含量亦较丰富。萝卜花粉资源丰富,营养成分齐全,具有很好的开发利用价值。     

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

ABSTRACT: BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.     

Interactions between elicitins and radish Raphanus sativus

Two responses to elicitins are described in cultivars of radish (Raphanus sativus L.). Type I, exhibited by the cultivar Daikon, is characterised by wilting and desiccation within 24 h of elicitin application and was previously reported as the sensitive response (S. Kamoun et al. 1993, Mol Plant-Microbe Interact 6: 15–25). At 1 μg elicitin · g⁻¹ FW radish tissue, symptoms appeared after 8 h, a sensitivity comparable to that shown by tobacco to β elicitins (J.-C. Pernollet et al., 1993, Physiol Mol Plant Pathol 42: 53–67; S. Kamoun et al., 1993, Mol Plant-Microbe Interact 6: 15–25). Elicitin failed to induce these symptoms in the cultivar White Icicle, even at 100 μg · g⁻¹ FW of tissue. However, a different response (Type II) with symptoms resembling senescence appeared in White Icicle after 48 h and were fully developed by 72 h. The Type II response was induced at levels of elicitin above 0.3 μg · g⁻¹ FW. Elicitin-treated Daikon leaves held at 100% relative humidity, rather than ambient (50–60%) did not wilt and by 72 h displayed Type II symptoms. When treated Daikon leaves were removed to ambient humidity at any time during the latent period, they developed Type I symptoms within 2 h. Although Type I symptoms were suppressed in Daikon at high humidity, there was no indication that leaf diffusion resistance or plant water conductance were affected. Protoplasts from the cultivar Daikon responded to elicitin by H⁺ uptake and K⁺ release, with maximal response at 300 pM. The response was eliminated by K252a or staurosporine. Daikon protoplasts also showed transient uptake/secretion of Ca²⁺ on elicitin addition. Protoplasts from White Icicle gave neither of these responses. Both Daikon and White Icicle phenotypes could be transferred to progeny of Daikon-White Icicle crosses and in the F2 generation three phenotypes, including a null, segregated. Only those F2 plants which exhibited the Daikon phenotype produced protoplasts which responded to elicitin. Received: 13 May 1997 / Accepted: 27 August 1997     

Changes in thiocyanate content in some radish Raphanus sativus L. cultivars during hypocotyl-root growth

Changes in the thiocyanate content in hypocotyl-roots and leaves of radish were observed in a two-year field experiment. Six cultivars were tested: early radish (Rex and Ostergruss Różowa), Japanese radish (Tokinashi and Minowase Summer Cross F₁), and winter radish (Monachijska Biała and Murzynka). A significant diversification in thiocyanate content among cultivars, plant parts, harvest dates and observation years was found. Early cultivars contained the least amount of these compounds, Murzynka — the greatest. The content of thiocyanates in leaves was 3 – 5 times higher than that in hypocotyl-roots. The changes in the thiocyanate content during root growth showed a constant rising tendency in the case of the leaves of all cultivars and the storage organs of Murzynka.     

Characterization of Radish (Raphanus sativus) Storage Proteins

Radish (Raphanus sativus cv Rond rose à bout blanc Vilmorin) seeds, as other cruciferae oil seeds, contain two major types of storage protein aggregates which can be separated by gel filtration into 12 and 1.7 Svedberg fractions. These two fractions have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, and two bidimensional gel electrophoresis systems. These results were compared with those obtained with rapeseed storage proteins. Radish 12 Svedberg particles are made of a series of nine major polypeptides ranging from 33 to 30 kilodaltons. These polypeptides present charge heterogeneity. The 12 Svedberg particle is made of six subunits 55 kilodaltons. Each subunit is a couple of two polypeptides linked by a disulfide bridge. The 1.7 Svedberg particle has a simpler composition. It is made of two polypeptides of 10 and 12 kilodaltons and smaller peptides of 7 kilodaltons. Twelve and 1.7 Svedberg particles also differ in their amino acid composition, the 1.7 Svedberg being particularly rich in glutamic acid and proline. Its components are basic. The organization of the rapeseed storage protein is similar but more complex.     

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

Non-Random Mating in a Wild Radish, Raphanus sativus

Abstract When mating is non-random among several, compatible donors, the fitness of pollen donors, maternal plants, and offspring may be affected. Although this process may be important, it is much less studied than other forms of non-random mating such as incompatibility and avoidance of inbreeding. Therefore, the amount and consequences of non-random mating were investigated in greenhouse studies with wild radish, Raphanus sativus . Six compatible donors differed in the number, position, and weight of seeds sired, so mating was non-random at the level of mate identity. Mate number also affected mating patterns; fruits with more fathers were allocated more resources. This keeps mate number per fruit high. In contrast, other processes appear to keep mate number below the maximum so that mate number per fruit is regulated at an intermediate level. Mate identity had clear consequences as offspring with different fathers were of different sizes after 11 weeks. The effects of mate number on offspring success were less clear. These and other data suggest that non-random mating among compatible donors is a relatively common process in wild radish. It may occur through mechanisms controlled by the pollen tubes, the maternal plants or the embryos. While this non-random mating is the raw maternal for sexual selection in plants, whether sexual selection actually occurs and how important it may be is still nuclear.     

Starch content of etiolated radish (Raphanus sativus L.) hypocotyls as affected by chloramphenicol and external sucrose

Abstract

Starch was enzymatically assayed in the hypocotyls of radish (Raphanus sativus L.) seedlings grown in water or chloramphenicol (CAP) 1 × 10^4. CAP inhibits starch formation and its effect is related to the concentration. Both CAP- and water-grown hypocotyls are able to accumulate starch when sucrose is supplied in the medium, thus suggesting that the damage caused by CAP to the amyloplast is not irreversible. Apical segments of both water- and CAP- grown hypocotyls accumulate starch upon incubation in sucrose solutions while basal segments are unable to accumulate starch even in the presence of sucrose. The authors suggest that the basal segments are unable to perform sucrose uptake or that the amyloplast is incapable to starch synthesis. In any case the inability of the basal segment to perform sucrose uptake is independent of CAP. These findings confirm that the radish hypocotyls is not homogeneous along its axes.     

Changes in growth regulator content of radish seedlings, Raphanus sativus, infested with the aphid Myzus persicae

Following 10 days of infestation by the aphid Myzus persicae there was an increase in the amount of growth inhibiting substances and a decrease in cytokinins, gibberellins and auxins in infested as compared with similar uninfested radish seedlings. Even after previously infested seedlings have been freed of aphids for 10 days, differences in the hormone balance remained. The possible relationships between the changes in hormonal balance and the effect of the aphid infestation on growth, translocation and wilting are discussed.     

Studies on Stigma Pellicle Glycoproteins of Raphanus sativus L.

Stigma surface diffusates of Raphanus sativus were subjected to polyacrylamide gel electrophoresis. There appeared protein bands, one of which gave positive PAS reaction, indicating it was glycoprotein. SDS polyacrylamide gel electrophoresis showed that the stigma diffusates contained many protein bands. By comparing their mobilities with those of standard proteins of known molecular weights, the molecular weights of some of the major fractions were estimated to be 15000, 30000—46000 and 70000 daltons. After Schiff reagent staining two main glycoprotein fractions appeared on the SDS gel electrophoretic pattern. Their molecular weights were estimated to be lower than 15000 and higher than 100000 daltons. By using acrylamide gel isoelectric focusing method, it was found that the stigma surface diffusates contained an acidic glycoprotein with pH of about 3.7. The amino acid composition of the purified stigma glycoprotein was determined with amino acid analyzer. Glycine, glutamic acid, serine, aspartic acid were some of the predominant amino acids. The diffusates were analysed by gasliquid chromatography for sugars. Results showed that the carbohydrate fraction of the glycoprotein consisted of arabinose 17.3%; galactose 19.1%, xylose 8.1%, mannose 5.4%, glucose 23.7%, rhamnose and/or fucose 26.4%. In the stigma surface diffusates of Raphanus sativus, the content of protein was estimated to be 16% and that carbohydrate was 11%.